Protein-protein cross-linking and membrane lipid oxidation are important features of oxidative pathways that lead to erythrocyte lysis and myocardial reperfusion injury. These deleterious processes are promoted by the reactions of hemoglobin or myoglobin with H2O2 and other redox active agents. Oxidative damage to these and other hemoproteins increases both normal heme catabolism by heme oxygenase and abnormal heme degradation. The purpose of this project is to define the chemical mechanisms of the interactions of hemoglobin and myoglobin with peroxides, to explore the relationship of these reactions to protein cross-linking and membrane damage, and to characterize the normal and abnormal pathways of heme catabolism. In the previous period of support, the focus of the project was on damage to myoglobin by H2O2 and other agents. In the proposed period of support, the focus is shifting to some extent towards heme oxygenase and abnormal heme catabolism. The immediate goals are to: (a) complete identification of the amino acid(s) that are oxidized in myoglobin in the reaction with H2O2, (b)express human heme oxygenase isoform-2 (HO-2) by the methods used to express isoform-1 (HO-1), (c) elucidate the structure of human HO-I by spectroscopic and physical methods, (d) determine the mechanism(s) of human HO-1 and HO-2, (d) explore the mechanism of the abnormal H2O2-dependent degradation of heme groups to monopyrrole and dipyrrole products, and (e) investigate the relationship of heme oxygenase catalysis to the catalytic processes supported by other types of hemoproteins. The results should help to clarify the molecular aspects of the oxidation of myoglobin and hemoglobin by peroxides and the mechanisms of both normal and abnormal heme catabolism. These results are relevant to oxidant-dependent hemolysis and reperfusion injury, oxidative hemoprotein damage, and the putative role of brain HO-2 as part of a neural messenger system.